Fetal DNA shed from the placenta is detectable in maternal blood at levels between 3% and 6% of total DNA. Thus, a number of PCR based fetal genetic screens have been described (e.g., gender, Rh, and thalassemia. However, traditional techniques are limited for at least two primary reasons: first, PCR assays trade sensitivity for specificity, often making it difficult to identify particular mutations, and second, aneuplodies, such as Down's Syndrome, cannot be detected by conventional PCR in a heterogeneous sample.
One way to avoid the problems of PCR-based assays is through large amounts of DNA using highly scalable techniques. Such digital analysis approaches involve the separation of extracted genomic material into discrete subsamples so that target sequence detection is binary (0, 1). For example, digital PCR allows amplification of single molecules, followed by quantitative analysis. Digital PCR typically requires limited dilution of a nucleic acid in a multi-well format followed by amplification of nucleic acids in the wells.
Digital PCR has been applied to fetal diagnostics in order to detect fetal mutations with specificity and sensitivity beyond that of conventional PCR. However, such methods are optimized for analyzing a single target in a biological sample, and moreover, the sample is consumed without interrogating the identity of other targets in the sample. Furthermore, digital PCR is limited to optical detection. Thus, multiplex analysis using digital PCR is not feasible, as it would be difficult to distinguish between optical signals for multiple targets in a given well. Additionally, conventional digital analysis does not allow for the detection of fetal single nucleotide polymorphisms (SNPs), which could be informative regarding a broad spectrum of diseases or conditions in the fetus. Thus, there is a need for improved methods for non-invasive, prenatal screening for multiple genotypes and/or mutations on many different targets genes at the single nucleotide level.